Utility of molecular studies in incontinentia pigmenti patients

The diagnosis of incontinentia pigmenti (IP) is fairly easy in the presence of classical features, but can be difficult in cases with partial or non-classical features, especially in the parents. The demonstration that the disease is caused by mutations in the NEMO gene, has remarkably improved genetic counselling for this disorder. We present four families of IP in whom molecular studies established an unequivocal diagnosis in the affected daughters, and showed two mothers to be carriers, thus allowing accurate genetic counselling and prenatal diagnosis.

severe compared to those causing ED-ID and OL-ED-ID. Mutation analysis of NEMO gene confirms the clinical diagnosis and is necessary for prenatal diagnosis in future pregnancies. The study was done on four consecutive IP patients who presented to us for genetic counselling or prenatal diagnosis during 2005-2008. Case 1: A 33-year-old second gravida consulted at 7 weeks of pregnancy. Her first child was a 12 yr old daughter who had short stature (height 142 cm), with hypopigmented skin lesions along the Blaschko lines all over the body. Blisters were present at birth. Biopsy of these lesions showed different stages of skin lesions consistent with incontinentia pigmenti. There was no vision in the left eye due to retinal detachment at the age of 1.5 yr. Nails were dystrophic in both hands and feet. She had partial anodontia-total teeth 18. The mother had short stature and she also had partial anodontia with minimal skin lesions. Her nails and hair were normal with no eye lesions. Both mother and daughter did not have any CNS manifestation.
The grandmother of index case was also short and had abnormal teeth. There was history of death of male children soon after birth in grandmother's sister. Molecular studies confirmed the diagnosis in both mother and her daughter and prenatal diagnosis was provided in her ongoing pregnancy.
Case 2: A 2 yr old girl with hypopigmented skin lesions had blisters present at the time of birth. Skin biopsy at day 9 suggested IP. Her eyes, teeth and nails were normal. There was no mental retardation. Examination of parents for any skin lesions, nail or hair changes was negative. There was no history of other affected females or recurrent foetal losses. Molecular studies established the diagnosis in the affected child, whereas the mother was shown not to be a carrier.
Case 3: A 4 yr old girl born to nonconsanguineous parents, had hyperpigmented skin lesions along Blaschko lines at the time of presentation (Fig. 1). She had blisters present at the age of one week. Skin biopsy suggested stage 2 of IP. She had oligodontia. Her eyes and nails were normal. Examination of parents for any skin lesions, nail or hair changes was negative. Molecular studies confirmed the diagnosis in the daughter, whereas the mother was excluded as a carrier.

Case 4:
This family had come for prenatal diagnosis of IP as their 5 yr old daughter was clinically suspected to have IP. She had hypopigmented streaks in whorled pattern but not along Blaschko lines. These hypopigmented lesions were preceded by rough papules. There was no history of vesicles at birth. The child had peg-shaped teeth. She had history of seizures. Her mother had atypical hypopigmented skin lesions. Both mother and daughter had the deletion of NEMO gene thus confirming the diagnosis.
Clinical features of cases are summarized in the Table. Molecular diagnosis of incontinentia pigmenti: DNA was isolated from the peripheral blood from all affected girls and their parents. The deletion in NEMO gene was analyzed by two PCR reactions according to the protocol described earlier 6,7 . Multiplex PCR as described by Steffann et al 6 was done first to detect deletion in NEMO or pseudogene. In the method, the internal control of amplification is present which rules out any PCR artifact when rearrangement of NEMO or pseudogene is absent, but this test cannot discriminate between NEMO and psudogene rearrangement. So, the PCR method described by Bardaro,et al 7 to distinguish between NEMO and psudogene rearrangement was done.
PCR showed that the index case in all the families had a band corresponding to the deletion of exon 4-10 in NEMO gene (1045 bp; Fig. 2). The mother in the cases 1 and 4 also had the same deletion whereas parents were normal in other two families.
Diagnosis of IP is made by the diagnostic criterion given by Landy and Donai 2 . Significant clinical heterogeneity exists in IP with regard to ectodermal, ophthalmologic, and neurologic abnormalities, even within families. Skin abnormalities are consistent  IP is familial in 10-25 per cent of cases. In this study, two cases had affected mother whereas parents were normal in the remaining two cases. IP is an xlinked dominant disorder and the risk of recurrence depends upon mutation status of parents. If parents have a deletion in the NEMO gene, risk is 50 per cent to all offsprings (male and females). Affected male children do not survive beyond second trimester. If parents do not have deletion the risk in next pregnancy is low (about 1%).
Multiplex PCR of the NEMO gene was done first which showed 1045 bp band in all four cases. This confirmed the clinical diagnosis in all the four cases. By this multiplex PCR, rearrangement of both NEMO gene and pseudogene is detected. To exclude pseudogene rearrangement PCR method described by Bardaro,et al 7 was done in all cases. All four cases had deletion in NEMO gene. These tests detect the common rearrangement as recurrent exon 4-10 genomic rearrangement in the NEMO gene accounts for 60 to 80 per cent of IP-causing mutations 10,11 .
Prenatal as well as postnatal diagnosis is best done by the combined use of molecular tests 6,7 to detect the deletion and direct sequencing of the gene, if deletion is absent.
Most of the studies on IP are reported from western countries 12 . A few reports of IP are available from China, Korea and Japan 12 . In general, the frequency of NEMO mutation is similar 12 . Besides the NEMO rearrangement found in IP females (which is lethal in males), a total of 69 different small mutations (missense, frameshift, nonsense, and splice-site mutations) have been reported, including 13 novel ones 11 . Hence, if deletion is absent sequencing of NEMO gene would be required. x-inactivation studies may help further.
In conclusion, clinical features of IP are variable and difficult to diagnose in cases with mild manifestations. Molecular diagnosis may help confirm the clinical suspicion and is essential for providing definite genetic counselling and prenatal diagnosis.